Soybean proteins have been widely utilized for improving physical properties of foods because of their peculiar gelling property, and at the same time, use of soybean proteins as highly nutritious health food materials has been increased.
A storage protein of a soybean is precipitated at around pH 4.5, and therefore soybean proteins can be relatively simply separated into an acid-soluble protein fraction mainly containing soluble components other than a storage protein and an acid-precipitable protein fraction mainly containing a storage protein. The acid-precipitable protein fraction is collected to obtain an isolated soybean protein, which has been currently widely used in the food industry.
Soybean proteins are classified into 2S-globulin, 7S-globulin, 11S-globulin and 15S-globulin based on sedimentation coefficient by ultracentrifuge analysis. Among them, 7S-globulin and 11S globulin are the main constituent protein components of a globulin fraction. Herein, β-conglycinin and glycinin according to immunological nomenclature substantially correspond to 7S-globulin and 11S-globulin respectively.
Soybean proteins are each different in physical properties such as viscosity, coagulation property and surface activity, and nutritional physiological functions.
For example, it has been reported that 7S-globulin decrease neutral fat in blood (see Non-Patent Document 1). It has been believed that 11S-globulin has a high gelling activity and governs the hardness and mouth feeling of a bean curd gel.
Thus, a fractionation of soybean proteins into fractions rich in these components enables physiological functions or physical properties peculiar to each protein component to greatly manifest, probably leading to creation of a characteristic material. Therefore, extension of the field utilizing soybean protein in the food industry can be expected.
As seen from FIG. 1 showing dissolution behavior of 7S-globulin and 11S-globulin for a pH, 7S-globulin has a low solubility at about pH 4.8 and 11S-globulin has a low solubility at from pH 4.5 to 6. Thus, it is expected that respective components with a high purity may be fractionated by first precipitating 11S-globulin at about pH 6 and then further lowering pH to precipitate 7S-globulin.
However, in fact, when soybean milk is adjusted to pH 6 and separated into an insoluble fraction and a water-soluble fraction, and then, these fractions are subjected to SDS-polyacrylamide gel electrophoresis, the obtained electrophoresis patterns show that considerable amounts of 7S-globulin and 11S-globulin are mixed in both fractions.
For this reason, there is a problem that only a simple fractionation based on dissolution behavior of both globulins for a pH can not obtain their fractions with a high purity.
In order to overcome such a problem, some techniques for fractionation of 7S-globulin and 11S-globulin have been disclosed (see Non-Patent Document 2, Patent Documents 1 to 7 etc.).
On the other hand, it has been reported in recent years that acid-precipitable soybean proteins comprise various proteins having high affinity for polar lipids which constitute the membrane of a protein body, an oil body and the like including a cell membrane, in addition to 7S-globulin and 11S-globulin (see Non-Patent Document 3).
Considering such a report, the present inventors have studied. As a result, they have found that when sodium sulfate is added to low-denatured defatted soybean milk so as to be a 1M concentration and the soybean milk is then adjusted to pH 4.5 with hydrochloric acid, 7S- and 11S-globulins transfer into an acid-soluble fraction and other various proteins transfer into an acid-precipitable fraction (see Non-Patent Document 4).
It has also been found that the nitrogen amount of the acid-precipitable fraction accounted for about 30% of the total nitrogen amount in the defatted soybean milk, which is an unexpected large amount.
Further, it has been reported that industrially produced isolated soybean proteins contain about 35% of these various proteins, and it has been found that such a group of proteins influence the flavor of conventional soybean protein materials such as soybean milk or isolated soybean protein (see Non-Patent Document 5).
The acid-precipitable fraction that is not rich in 7S-globulin and 11S-globulin comprises proteins having mainly a deduced molecular weight of 34 kDa, 24 kDa and 18 kDa based on SDS-polyacrylamide electrophoresis, lipoxygenase, γ-conglycinin and other many various proteins.
Such a group of proteins have affinity for a polar lipid, therefore, they are called lipophilic proteins.
According to the above-described findings, it is understood that the previous fractionation techniques (Non-Patent Document 2, Patent Documents 1 to 7) can not substantially attain fractionation of 7S-globulin and 11S-globulin with a high purity because it is not considered at all that lipophilic proteins account for a considerable proportion of an acid-precipitable soybean protein fraction.
Although Non-Patent Document 4 has shown a method for fractionation of 7S-globulin, 11S-globulin and a lipophilic protein with a high purity, said method needs use of a large amount of a reducing agent under a high ionic strength and thus needs a desalting step and a washing step. Therefore, said method is effective at the experimental level, but is not suitable for an industrial process.
Then, the present applicant has developed a technique for fractionating soybean proteins into a soybean 7S-globulin protein fraction with a high purity which has a low content rate of a lipophilic protein, and a soybean 11S-globulin protein fraction (see Patent Documents 8 and 9). Said method has been industrially excellent in that 7S-globulin with a high purity can be fractionated. However, on the other hand, a troublesome procedure is needed in order to fractionate a mixture of 11S-globulin and the lipophilic protein, which is the remaining fraction, into each component with a high purity. Therefore, it has been in the situation that these components are not effectively utilized.
That is, not a method for fractionation of only 7S-globulin with a high purity, but a simple method for fractionation of the remaining fraction with a high purity is desired.
(Reference Documents)
Non-Patent Document 1: Okita T et al., J. Nutr. Sci. Vitaminol., 27(4), 379-388, 1981
Non-Patent Document 2: Thanh, V. H, and Shibasaki, K., J. Agric. Food Chem., 24, 1117-1121, 1976
Non-Patent Document 3: Herman, Planta, 172, 336-345, 1987
Non-Patent Document 4: Samoto M et al., Biosci. Biotechnol. Biochem., 58(11), 2123-2125, 1994
Non-Patent Document 5: Samoto M et al., Biosci. Biotechnol. Biochem., 62(5), 935-940, 1998
Non-Patent Document 6: T. Nagano, et al., Relationship between rheological properties and conformational states of 7S globulin from soybeans at acidic pH, Food Hydrocolloids: Structures, Properties, and Functions, Plenum Press, New York, 1994
Patent Document 1: JP 55-124457 A
Patent Document 2: JP 48-56843 A
Patent Document 3: JP 49-31843 A
Patent Document 4: JP 58-36345 A
Patent Document 5: JP 61-187755 A
Patent Document 6: WO 00/58492 A1
Patent Document 7: U.S. Pat. No. 6,171,640 B1
Patent Document 8: WO 02/28198 A1
Patent Document 9: WO 2004/43160 A1